1. Field of the Invention
The present invention relates to a system and method for the infiltration of plants in a continuous or quasi-continuous operation mode. The system and method of the present invention combine mechanical (automatic and manual) movable and manipulable units and/or containers with vacuum infiltration chambers to enable large-scale infiltration of plants.
2. Description of Related Art
Genetic transformation of higher plants promises to have a major impact on large-scale production of proteins of industrial importance. Various methods are known to modify plants by genetic transformation. One approach to produce foreign proteins in plants is to generate stable transgenic plant lines. A faster alternative method is based upon transient transformation by contacting the surface of a plant with a suspension of infectious particles such as a virus and/or microorganisms such as agrobacterium tumefaciens. By the infiltration of the infectious particles and/or microorganisms, the genetic information is transiently introduced into the cell.
The infiltration can for example be realized by using the magnICON™ technology [EP1616959]. Here, the plants, growing in solid substrate, are dipped upside down in a suspension of agrobacteria in buffer. In this state a vacuum is drawn leading to a degasification of the plants capillaries (predominantly in the leafs). After the vacuum is reached, it is released rapidly so that agrobacteria suspension is drawn into the plants capillaries. The agrobacteria can now infect the plant cells very efficiently leading to a transient genetic modification of the plant. The plant is taken out of the suspension; turned again upside up and brought to the incubation area. Here, the modified plant cells are producing the specific protein. After incubation the plants are harvested and the protein is purified. However, this method has so far only been conducted on a laboratory scale.
Conventional devices for carrying out the vacuum infiltration method are for example box type chambers with a top lid. The box is filled with an agrobacteria suspension and the plants are placed upside down. The lid is closed and the vacuum is drawn. Beneficial in this approach is predominantly the nearly optimal usage of space. On the other hand the open handling of the plants after the dipping process is likely to cause spilling not only on the vacuum chamber itself, but also on the handling unit and the room. As the agrobacteria suspension contains genetically modified organisms (GMO's), spilling has to be avoided. If spills can not be prevented, the device should at least be easy cleanable. Both problems are not addressed by conventional box type vacuum chambers.
Another issue with such conventional devices is scalability. The dimensions of a conventional box type chamber are illustratively in width: 1-2 m; length: 1-2 m; and height: 1-2 m. For a very large dipper unit, a massive and expensive scale-up by numbering would be necessary.
Infiltration of plants with or without vacuum is a known bench-scale method [see e.g. Clough et al., The Plant Journal (1998) 16(6), 735-743]. The method is normally performed with entire plants. Sometimes only some parts of the plant are cut off and processed (such as e.g. a leaf). In either case, the manipulation of plants or part of plants are carried out by hand.
In chemistry, exsiccators/drying chambers or small vacuum chambers are normally used as vacuum chambers. These are either directly filled with a liquid comprising GMO's or poured into a cartridge (inliner).
A large-scale use of vacuum chambers for plant infiltration is not known. A plant manipulation method which uses vacuum chambers is the boiler-vacuum-pressure method (full pressure method) for the impregnation of wood [e.g. Bayerisches Landesamt für Wasserwirtschaft, Merkblatt Nr. 3.3/3 from Mar. 21, 1995]. With this method, stacked wood is transferred batchwise into a vacuum chamber. The vacuum chamber is closed; vacuum is drawn and the chamber is flooded with an impregnation agent. After vacuum is removed, the impregnation agent infiltrates the wood and the remaining impregnation agent is drained. Then, the vacuum chamber can be opened again.
Due to various reasons this method is not commensurate with the solution provided by the present invention. Flooding of the chamber cannot be realized with liquid comprising GMO's as the chamber can be contaminated and the environment can be polluted after the chamber is opened. In addition, with this method the plant substrate would be soaked with liquid comprising GMO's. However, due to hygienic reasons and the fact that an unproportional high amount of liquid comprising GMO's would be lost, this has to be avoided. Furthermore, the solution provided by the present invention requires applying the vacuum after dipping of the plants. The described boiler-vacuum pressure method does not allow such a chronological order.
A further difference to the boiler-vacuum-pressure method results from the low throughput per batch due to the fact that plants—in contrast to wood—are to sensitive for stacking. The boiler-vacuum pressure method is also not suited as the process time for one batch is several hours. Such a high residence time is adverse for a high-through put approach. The solution provided by the present invention allows infiltration of plants in a continuous or quasi-continuous operation mode with cycle times of 20 minutes or lower. A technical operation mode with more than one unit in a continuous or semi-continuous way was not known in the prior art.